U.S. patent number 6,258,325 [Application Number 08/441,794] was granted by the patent office on 2001-07-10 for method and apparatus for preventing cross-contamination of multi-well test plates.
Invention is credited to Ashok Ramesh Sanadi.
United States Patent |
6,258,325 |
Sanadi |
July 10, 2001 |
Method and apparatus for preventing cross-contamination of
multi-well test plates
Abstract
A multi-well plate which prevents cross-contamination of samples
through the use of a resilient gasket which covers a majority of
the top of the plate and is compressed by a lid having a clamp
assembly. It thus provides a sealing assembly for arrays of
containers of any size or shape. The gaskets may be unitary sheets
with or without an array of openings corresponding to the well
openings or may consist of discrete single well gaskets. A
multi-tube array is also provided which can be sealed without the
need of a gasket or tight-fitting caps. A multi-well plate is also
disclosed in which samples can be gas-equilibrated without the risk
of microbial contamination.
Inventors: |
Sanadi; Ashok Ramesh
(Arlington, VA) |
Family
ID: |
26726916 |
Appl.
No.: |
08/441,794 |
Filed: |
May 16, 1995 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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195125 |
Apr 4, 1994 |
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049171 |
Apr 19, 1993 |
5342581 |
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Current U.S.
Class: |
422/549; 422/553;
436/177; 436/178 |
Current CPC
Class: |
B01L
3/5025 (20130101); B01L 3/50255 (20130101); B01L
3/5085 (20130101); B01L 3/50853 (20130101); Y10T
436/25375 (20150115); Y10T 436/255 (20150115) |
Current International
Class: |
B01L
3/00 (20060101); B01L 011/00 () |
Field of
Search: |
;422/58,69,101,102,104
;436/165,177,178,809 ;435/301,311 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0311440A2 |
|
Oct 1988 |
|
EP |
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0388159A2 |
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Mar 1990 |
|
EP |
|
Other References
Brochure of the Beadprep 96, Magnetic Separator/Concentrator. .
Millititer Filtration System, Bio/Analytical Test Products. .
96, 192, and 384 Well Plates For Every Occasion, USA Scientific
Plastics, Inc., 1993..
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Primary Examiner: Soderquist; Arlen
Attorney, Agent or Firm: Dykema Gossett PLLC
Parent Case Text
TECHNICAL FIELD OF THE INVENTION
This is a continuation of application Ser. No. 08/195,125 filed on
Apr. 4, 1994.
This application is a continuation-in-part of application Ser. No.
08/049,171, filed Apr. 19, 1993 now U.S. Pat. No. 5,342,581
entitled "Method and Apparatus For Preventing Cross-Contamination
of Multi-Well Test Plates" the entire disclosure of which is
incorporated herein by reference.
Claims
What is claimed is:
1. A specimen containment assembly, comprising:
a lid;
a plate defining a plurality of individual sample containment
chambers, said chambers each having an open end and a closed end,
said closed ends and said plate being formed of a substantially
fluid impermeable material;
a fluid impermeable gasket interposed between said lid and said
plate;
said lid defining a plurality of openings in register with said
open ends of said chambers to provide access to said chambers;
and
a clamp for clamping said lid, plate and gasket together such that
said gasket is compressed between said lid and plate;
said lid, plate, and gasket cooperating to hermetically seal said
individual sample containment chambers.
2. The specimen containment assembly recited in claim 1, wherein
said plate defining sample containment chambers is an array of
tubes.
3. The specimen containment assembly recited in claim 1, wherein
said plate is a multi-well titer plate.
4. The specimen containment assembly recited in claim 1, wherein
said gasket is compressible.
5. The specimen containment assembly recited in claim 1, wherein
said gasket is a self-sealing membrane having the capacity to
self-seal after removal of a syringe which pierces said
self-sealing membrane to access said sample containment site.
6. The specimen containment assembly recited in claim 1 further
including a fluid impermeable membrane interposed between said lid
and said plate.
7. The specimen containment assembly recited in claim 6, wherein
said gasket has a plurality of openings in register with said
openings in said lid.
Description
This invention relates generally to multi-well plates and tube
arrays in which samples (biological, chemical, etc.) are analyzed
or processed. More specifically, the present invention solves the
problem associated with cross-contamination of samples which may
occur in the use of a closely spaced array of wells or tubes. The
present invention also relates to a multi-well plate which can be
used for analysis or processing in a controlled atmosphere without
the possibility of continuation from atmospheric sources. In
addition, the present invention describes a new type of multi-well
plate which can be clamped together.
BACKGROUND OF THE INVENTION
A number of laboratory and clinical procedures require the use of
an array of wells or tubes in which multiple samples are placed for
analysis, cell growth, amplification, isolation or other purposes.
In general, conventional multi-well plates and tube arrays
(non-filtration type) have a single opening at the top through
which samples are added or removed.
An important disadvantage in the use of arrays of tubes mounted
within a plate, and with multi-well plates (either with or without
a filtration feature) is the problem associated with contamination
of the samples. Most laboratory protocols must be performed with a
high degree of stringency in terms of limiting contamination of the
samples. When multiple samples are processed in a confined area,
such as an 8.times.12 or 4.times.6 format, strip wells (in strips
of 8 or 12 wells), or in any format with multiple sample containers
in a small area, the risk of cross-contamination of samples is
significant, giving rise to erroneous results. If a single unitary
plate (as currently available) is used as a collective lid to cover
the tops of all the wells or tubes, the lack of a seal could allow
the migration of samples between wells or tubes during handling, or
simply through condensation and capillary processes. Tapes which
are used currently to seal the tops of the wells are not very
reliable. Adhesive tapes limit the number of conditions that the
plate can be subjected to (efficient boiling, freeze-thawing and
vortexing the plate are difficult without causing
cross-contamination) and heat sealing tape requires specialized
heat-sealing equipment. Incorporation of a tape sealing process in
automated systems would be difficult. In addition, multi-well or
tube arrays which utilize individual stoppers are unwieldy and
allow the introduction of contaminants as the reagents and the like
are added to the wells/tubes. This problem of cross-contamination
is particularly acute when tight fitting caps and tape are opened,
which frequently results in aerosol formation. These aerosols, in
addition to being a potential source of cross-contamination, may
also be hazardous to the operator.
A problem often encountered with cell culture procedures is the
contamination of the cultures by microorganisms from the
environment or the atmosphere. This problem has been difficult to
overcome, because cell-culture procedures often require the
microorganisms to be grown in, a controlled atmosphere (such as 5%
carbon dioxide); the conventional plates therefore have a loose
fitting lid to reduce evaporation while allowing gas exchange and
yet minimizing contamination. Also, it would not be possible to
clamp the lid to the multi-well plate without changing the
dimensions of the plate, which would make it difficult to use with
existing instruments such as plate readers, centrifuges and the
like. It is important to appreciate that the use of the membrane in
the present invention is very different from prior art involving
96-well filtration devices, where the liquid samples have to come
in contact with the membrane for the purpose of filtration. Thus,
in the prior art, the membrane provides for flow-through of liquid,
with the liquid often in contact with the membrane for prolonged
periods of time prior to filtration. In the present invention the
membrane prevents flow-through of non-gaseous materials, but allows
gas-exchange.
Conventional glass microscope slides having one or more wells are
now being used as sample holders for in situ nucleic acid
amplification techniques such as PCR. Generally, either glue or
cosmetic nail enamel is used to stick the cover directly to the
slide, requiring the use of heat or a solvent to remove the
cover.
Therefore, it is an object of the present invention to provide a
plate/tray assembly having an array of sample containment site,
which are designed to reduce the risk of cross-contamination
between containment sites.
It is another object of the present invention to provide a
multi-well plate or tube array in which cross-contamination of
samples is significantly reduced by providing a resilient gasket
which isolates each containment site.
It is yet another object of the present invention to have a tube
array (or multi-well plate) which can be sealed without the use of
a gasket or tight fit caps.
It is a further object of the present invention to provide a method
of leaving samples in the sample containment sites in the
multi-well plate/tube array open to the atmosphere and yet sealed
from microbial, particulate or other contamination from atmospheric
sources.
It is still a further object of the present invention to provide a
sample containment assembly of multiple samples (such as 96 well
plates and cluster plates) which can be hermetically sealed and
clamped together without changing the effective dimensions of the
assembly so that standardized equipment such as automated well
washers, automated scanning instruments and centrifuges can still
be used.
Finally, it is still a further object of the present invention to
provide a sealing system for glass or plastic slides, which can be
used without gluing the cover slip to the slide.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides an apparatus for
handling multiple samples having a plurality of containment sites
such as wells or tube-like vessels defining wells. The wells or
tubes may be discrete elements temporarily attached to a tray or
plate or preferably are formed integrally with a plate. Each well
has one closed end and one open end. The plate (and thus the closed
end) may be formed of a number of fluid impervious materials such
as a rigid plastic. The apparatus further includes a lid which
covers the principal or top surface of the plate or tray such that
the lid simultaneously covers all of the openings of the
containment sites or wells. Between the lid and the principal
surface of the tray or plate, a layer of resilient material such as
a synthetic rubber membrane is provided which serves as a gasket.
In one embodiment the gasket is a single unitary sheet which covers
all of the openings of the containment sites of the plate or tray.
Thus, the gasket serves as a closure for each specimen chamber. The
lid is then clamped or otherwise secured to the plate or tray with
sufficient force to compress the gasket and provide sealing contact
between the gasket and the tray or plate to seal the well openings.
The apparatus can then be placed in various orientations without
movement or loss of the samples from their respective containment
sites.
In another aspect, the gasket feature of the present invention
comprises a plurality of discrete gaskets or gasket sections each
of which covers one or several openings of the plate. The discrete
gaskets extend beyond each individual opening a sufficient distance
to provide a seal between the individual containment sites.
In still another aspect, the gasket of the present invention is
further provided with openings in register or alignment with each
of the openings of the containment sites of the plate or tray such
that access to the individual containment sites may be achieved by
simply removing the lid.
In another embodiment, a mylar sheet or membrane is disposed on top
of the gasket in that embodiment in which the gasket has a
plurality of openings.
In yet another embodiment, a new format of multi-well plates or
tube arrays is provided which allows the securing of the lid to the
plate or tube array, without changing the dimensions of the
apparatus, so that current instrumentation for handling 96 well
plates can still be utilized.
In addition, a multi-well plate or tube array is provided which has
a plate defining a plurality of sample containment sites, each of
the containment sites having an internal shoulder or annular rim; a
gasket or O-ring disposed on the internal annular rim, and a lid
having a projection that mates with and compresses the gasket or
O-ring to seal the containment site.
The present invention also provides an apparatus that allows gas
equilibration of the samples in the containment sites with the
ambient atmosphere while preventing microbial, particulate, or
chemical contamination of the samples from the atmosphere.
In yet another embodiment, the present invention provides a design
whereby multiple sample containers can be sealed without the use of
tight fitting caps or a gasket.
Finally, the present invention provides a glass slide that can be
sealed without using any adhesives.
These and additional aspects of the present invention will be more
fully described in the following detailed descriptions of the
preferred embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an exploded perspective view of a multi-tube tray
assembly made in accordance with the present invention.
FIG. 1A shows a perspective view of the multi-tube plate of the
assembly shown in FIG. 1.
FIG. 1B shows a cross-sectional elevational view of the assembly
described in detail with reference to FIG. 1.
FIG. 2 is an exploded perspective view of a multi-well titer plate
assembly made in accordance with the present invention.
FIG. 2A is a perspective view of the apparatus described in detail
with reference to FIG. 2.
FIG. 3 is a cross-sectional elevational view of a multi-well plate
in one embodiment of the present invention.
FIG. 3A is an exploded perspective view of the invention described
in detail with reference to FIG. 3.
FIG. 3B is a plan view of the gasket depicted in FIG. 3.
FIG. 3C is a plan view of a thermal equilibration membrane with
annular rings of resilient gasket material fixed to it.
FIG. 4 is an exploded perspective view of a multi-well plate in
another embodiment of the present invention.
FIG. 4A shows a fragmentary cross-sectional elevational view of the
apparatus shown in FIG. 4, with a single well and corresponding
portion of the lid broken away from the rest of the assembly.
FIG. 4B shows a fragmentary perspective view of the plate described
with respect to FIG. 4, showing in detail the clipping
mechanism.
FIG. 5 is a fragmentary cross-sectional elevational view of one
sample containment chamber in a multi-container assembly having
individual gaskets in accordance with one aspect of the present
invention.
FIG. 5A is a fragmentary cross-sectional elevational view of one
sample containment chamber made in accordance with another aspect
of the invention shown in FIG. 5.
FIG. 5B is a plan view of an individual gasket for use in the
apparatuses shown in FIGS. 5 and 5A.
FIG. 6 shows a cross-sectional elevational view of yet another
embodiment of the present invention, with a single well broken
away.
FIG. 7 shows a fragmentary cross-sectional elevational view of
another configuration of the invention described in detail with
respect to FIG. 6.
FIG. 7A is an exploded perspective view of the present invention in
another aspect.
FIG. 8 shows an exploded perspective view of a multi-tube array
made in accordance with the present invention, which can be sealed
without the use of a gasket or tight-fitting caps.
FIG. 8A shows a fragmentary cross-sectional view of the tube array
and cap assembly described with respect to FIG. 8.
FIG. 9 shows a cross-sectional view of a microscope slide in yet
another embodiment of the present invention.
FIG. 9A shows an exploded perspective view of the invention
described with respect to FIG. 9.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Referring to FIG. 1 of the drawings, in one embodiment assembly 18
is shown with tube tray 3 having a plurality of tubes 4, only one
of which is shown in FIG. 1 for the sake of clarity. Each tube 4 is
provided with an opening or mouth 11. It will be appreciated that
tray 3 may be formed as an integral or single-piece structure
having tubes 4 or that tubes 4 may be formed as discrete units
which are subsequently attached to tube tray 3 either temporarily
or permanently. For example, tube tray 3 could comprise a plate
with plurality of openings in which tubes 4 are held in the nature
of a test-tube rack, with a holding or retaining plate being fixed
reversibly and temporarily to the plate thereby holding the tubes
in place. Tube tray 3 is preferably placed in tray carrier 16
having a principal surface 19 which mates with lower surface 20 of
tube tray 3. Portions of tubes 4 which extend below lower surface
20 are received through holes 17 of tray carrier 16 into bores 6 of
base plate 5. When tube tray 3 is inserted into tray carrier 16,
protrusions or retaining arms 7 on tube tray 3 will engage tray
carrier 16 at slot 8, thereby holding tube tray 3 in place. In this
particular embodiment, slot 8 in side wall 13 is in the form of an
inverted "T". When retaining arms 7 are first inserted into the
vertical portion of slot 8, retaining arms 7 are pushed closer
together by wall 13; when fully inserted into the final position,
retaining arms 7 will be engaged at the wider portion of slot 8,
returning to their normal positions by virtue of their flexibility,
since side wall 13 will no longer be pressing them closer to each
other. Thus, retaining arms 7 function as a snap-in fitting in this
embodiment. In some embodiments it may be necessary to manually
compress arms 7 together to slide them into position. Thus, tray 3
cannot be removed from carrier 16 without compressing arms 7
together in the direction shown by the arrows in FIG. 1A. It is to
be appreciated that arms such as 7 can be built in more than one
side of tube tray 3 and reciprocal slot 8 can be built in
corresponding places of tray carrier 16 during manufacture. In
FIGS. 1 and 1A, retaining arms 7 and slots 8 are shown on only one
side of assembly 18 for the sake of simplicity. Other variations of
arms 7 and slot 8 can be made in light of the teachings of the
present invention.
Following the insertion of tube tray 3 in tray carrier 16 by
fitting retaining arms 7 in slots 8, samples can be introduced into
tubes 4. Sealing layer or resilient gasket 2, generally having the
same geometry as principal surface 21 of tube tray carrier 16 is
placed on top of tray 3 such that it covers the majority of
principal surface 21, including most and preferably all of the
individual openings 11. In general, gasket 2 comprises a resilient
sheet or membrane which should be inert with respect to the samples
within tubes 4. Gasket 2 may be formed of any deformable and
resilient material and should not adhere or stick to principal
surface 21 nor lid 1. In the preferred embodiment, the material
from which gasket 2 is formed is substantially impermeable to
liquids and gases. Examples of materials that could be used are
silicon rubber, neoprene, and the like. Many foamed material,
particularly closed-cell foams, are particularly resilient and are
suitable. Gasket 2 may also include a coating of a highly inert,
relatively inflexible material such as Teflon, which may be applied
in a thickness which does not interfere with the resiliency of
gasket 2. Resilient gasket 2 should have sufficient resiliency such
that when it is compressed it forms a seal for the tops 22 of tube
openings 11. The thickness of gasket 2 is not critical, but should
be enough to form a seal when compressed by lid 1. Lid 1 is then
placed on top of gasket 2, such that snap-in clip 9 is engaged by
hole 10 in shoulder 15 of tray carrier 16. Through this engagement,
pressure is applied to gasket 2 so that it seals openings 11 (most
preferably hermetically) of tubes 4. In this embodiment, surface 12
of lid 1 is a simple planar surface i.e. a flat surface which
applies a substantially equal force over the entire surface of
gasket 2. In a different embodiment, annular collars protruding
downwards from lower surface 12 of lid 1 are in alignment with tops
22 of tubes 4 thus exerting pressure on gasket 2 to insure sealing.
In other applications, it may be desirable to bond gasket discs or
annular rings to the lid, and numerous methods of attaching the
preferred gasket materials to the lid will be known to those
skilled in the art. Thus as shown in FIGS. 1 and 1B, lid 1 is in
the nature of a "box-top" construction having walls 14 and inner
lid surface 12. Lid 1 fits over walls 13 of tray carrier 16 as best
shown in FIG. 1B. Gasket 2 is shown compressed by surface 12 onto
the top of tubes 4. In still another embodiment, the thickness of
tray 3 (dimensions A in FIG. 1A) is such that principal surface 21
is substantially flush or co-planar with the tops of walls 13 of
tray 16. This latter configuration is particularly preferred where
tray 3 is in the nature of a multi-well titer plate.
In one embodiment, which will be explained more fully in connection
with FIGS. 3B and 3C, gasket 2 may be provided with an array of
openings corresponding to the well or tube openings. Where gasket 2
has these openings, a sheet such as a mylar membrane may be
disposed on top of resilient gasket 2. Thus, even after the lid is
removed, the samples in the tubes remain covered with the mylar
sheet, therefore reducing the chances of contamination of
samples.
Referring to FIG. 1A, a significant portion of tubes 4 extend above
the principal surface 21 of tray 3, with the top surfaces 22 of
tubes 4 in a single plane parallel to principal surface 21. Thus,
when tray 3 is placed in tray carrier 16 and gasket 2 is disposed
on top with lid 1 clamped in place, top surfaces 22 of tubes 4 and
the lower principal surface 12 of lid 1 compress gasket 2 between
them, thereby sealing openings 11 of tubes 4. Upon completion of
the reaction/processing/analysis, clip 9 can be opened and lid 1
removed. It is to appreciated that upon opening of clip 9 and
leaving lid 1 in place, the pressure on gasket 2 is released. The
resiliency of gasket 2 will therefore allow opening of the seal
without the formation of aerosols which are formed upon opening of
tight fitting snap-type caps. The gasket can also be
non-compressible and still seal the wells. Such a gasket could be
made of flexible but substantially non-compressible polyethylene,
polypropylene and the like. Such a gasket could be also bonded to
the lid, or the lid could be made of more than one material such
that a gasket is not required.
Referring now to FIG. 2 of the drawings, in another embodiment of
the present invention, assembly 39 has multi-well plate 23 provided
with sample containment wells 24, each well 24 having a well
opening 25 at principal surface 26. In the preferred embodiment,
the tops 37 of wells 24 are coplanar, and are raised above
principal surface 26 to facilitate sealing of wells 24 by gasket
30. It will be appreciated that multi-well plate 23 may comprise
flat-bottom, U-bottom, conical bottom or semi-circular wells or the
like. In this embodiment of the invention, plate 23 has a shoulder
27 which is wide enough to accommodate a clip or clamp assembly. It
will be appreciated that in the embodiment where plate 23 is a
conventional multi-well plate having an array of 96 wells, the
length L and width W (shown in FIG. 2A) of plate 23 will be
identical to the corresponding dimensions of conventional
commercially available 96 well plates. Resilient gasket 30 is also
provided which again covers principal surface 26 of plate 23 in
close contact therewith such that it seals wells 24 by covering
well openings 25 when lid 32 is snapped in place. Thermal
equilibration membrane 31 is shown disposed on resilient gasket 30
to provide rapid thermal equilibration if necessary. Thermal
equilibration membrane 31 will preferably be used in that
embodiment of the invention which includes a gasket having an array
of corresponding openings such as the gasket shown in FIG. 3B. It
will be appreciated that holes 46 of the gasket 45 shown in FIG. 3B
are in alignment with the well openings of principal surface 26. It
should be understood that a thermal sheet of this type will not be
necessary in many applications and will not be needed where the
gasket does not have an array of openings. Lid 32 is again provided
which serves to compress gasket 30 onto principal surface 26. Lid
32 is shown having a flat shoulder 33 which has a protruding ridge
34. When lid 32 is placed on plate 23 with gasket 30 in between,
and pressure applied to the top of lid 32, ridge 34 will be engaged
by the corresponding groove 35 in vertically protruding male member
36 on shoulder 27 of plate 23. Thus, it will be understood that
these structures are somewhat flexible to allow the necessary
bending for ridge 34 to be fitted into groove 35; that is to snap
into place. Gasket 30 will thus be compressed between lid 32 and
the tops 37 of well openings 25 thereby sealing wells 24 in the
manner previously described. It will be appreciated that upon
removal of lid 32, wells 24 will still be covered by gasket 30. The
complete assembly with lid 32 in place on plate 23 is shown in FIG.
2A as assembly 38. On applying pressure to the male member 36 in
the direction of the arrows as shown in the figure, groove 35 will
disengage ridge 34 thereby resulting in opening of the seal. It
will be appreciated that in this embodiment, male member 36 is
shown as an integral part of plate 23. However, male member 36
could also be an independent piece inserted into plate 23 for
holding lid 32 on plate 23 and sealing wells 24. Such variations of
the clipping or clamping mechanism could also include a snap,
hinge, sliding catch, or a hook, and will be apparent to those
skilled in the art in light of the teachings of the present
invention. In another embodiment (not shown), lid 32 has apertures
corresponding to well openings 25 on plate 23 so that samples can
be introduced into sample containment sites with a syringe or the
like through a resilient and self-sealing gasket without removal of
the lid. In the preferred embodiment, however, lid 32 does not have
holes. FIG. 2A shows an external perspective view of the assembled
invention described in detail with reference to FIG. 2. Being an
external view, gasket 30 and membrane 31 are not visible.
Referring now to FIG. 3 of the drawings, a multi-well assembly 41
made in accordance with the present invention is shown in
cross-sectional elevational view having multi-well plate 42
containing wells 43 at principal surface 44. Resilient gasket 45
having apertures 46 corresponding to well openings 47 is shown
disposed on plate 42 in the manner previously described. Lid 48
compresses resilient gasket 45 onto principal surface 44 of plate
42 to form a seal at regions 49 which, as will be recognized, are
those areas of principal surface 44 which surround each well 43. In
order to secure lid 48 and gasket 45 in place on multi-well plate
42, clamp 50 is shown, which in this embodiment is a simple
friction fit C-clamp or channel clamp. To hold the other side of
the lid 48 in place on plate 42 such that pressure is applied
uniformly to gasket 45, lip 51 is shown which slides into slots 52
in shoulder 53 of plate 42 in the nature of a tongue-in-groove
catch. Thus, to position lid 48 in place on gasket 45 which in turn
is disposed on plate 42, lid 48 is positioned at an angle to plate
42 so that lip 51 is engaged in slot 52 by shoulder 53 of plate 42.
The opposite (with respect to the side of lip 51) side 54 of lid 48
is then lowered into place and clamp 50 is put in place in notch 55
of plate 42 thereby sealing the well openings. The catch and clamps
may be of any convenient construction and may be attached to one or
more sides of the assembly as required. Variations of this design
will be apparent to those skilled in the art. It will be
appreciated that in use lid 48 may be covered with contaminants
such as dust, microorganisms or the like. In the present invention,
the lid can be removed prior to removal of gasket 45 in a sterile
or otherwise clean environment in those embodiments in which gasket
45 is bonded or otherwise interfaced within thermal equilibration
membrane 31. That is, wells. 43 will still be covered when lid 48
is removed by virtue of thermal sheet 31 overlying the
corresponding openings in gasket 45. The use and aforementioned.
modifications of thermal membrane 31 and resilient gasket 45 are
equally applicable to all embodiments of the present invention.
Multi-well assembly 41 is shown in perspective view in FIG. 3A;
note that gasket 45 and membrane 31 are not shown in FIG. 3A.
Referring now to FIG. 3B of the drawings, gasket 45 is shown having
openings 46 in alignment with wells 43. In this embodiment,
openings 46 have a diameter slightly smaller than the openings 47
of wells 43. This feature contributes to confinement of samples
within wells 43 to prevent cross-contamination. It will be
appreciated that by providing openings 46 in gasket 45, reagents
and the like can be easily added to wells 43 simply by removing
clamp 50 and lid 48 from the assembly 41 if membrane 31 is not
used. The lid and clamps can then be replaced to close and seal
wells 43. Alternatively, if the gasket has no openings, the lid can
have apertures in alignment with the well openings so that reagents
can be added by way of a syringe or the like, through the gasket,
the gasket being made in that embodiment of a self-sealing
material.
Referring now to FIG. 3C of the drawings, in an alternative
embodiment, the sealing function of the resilient gasket is
achieved by a modified thermal equilibration membrane or sheet 58.
Thermal equilibration sheet 58 has annular rings 59 made of
resilient material fixed or bonded onto it either by heat or
adhesive bonding, ultrasonic welding, or any desired means, which
would be well known to those skilled in the art. Thus, when sheet
58 is disposed between the lid and the base plate (having sample
containment sites), annular rings 59 are compressed between the
lower surface of the lid and the tops of the wells or tubes thereby
sealing the wells. In this embodiment, internal diameter 60 is of
slightly smaller diameter than the openings 47 of wells 43 as shown
in FIG. 3 which contributes to confinement of samples within wells
43 to prevent cross-contamination.
Referring now to FIG. 4 of the drawings, another embodiment of the
present invention is shown in exploded perspective view. Assembly
61 has a multi-well plate 62 having flat shoulder 63 and a
plurality of wells 64. Lid 65 has a shoulder 66 with holes 67 in
the shoulder. These holes are made such that when lid 65 is placed
on plate 62, male projections 68 are engaged by holes 67 in lid 65
and project through shoulder 66 of lid 65. Wells 64 have openings
whose tops 69 are preferably raised above principal surface 70 of
plate 62, making contact with the lower surface 71 of lid 65 when
lid 65 is placed in proper alignment on plate 62. After placing lid
65 on plate 62, clip 72, shown here as a flat, flexible friction
fit clip, is put in place such that lid 65 is held on plate 62,
with downward pressure being exerted by lower surface 71 of lid 65
on raised rims 69 of wells 64. An effective seal would thus be
formed by the mating of raised rims 69 and lower principal surface
71 of lid 65. Referring now to FIG. 4A, a single well 64 of
assembly 61 with collar 73 protruding from lover principal surface
71 of lid 65 is shown. Annular collars 73 protruding down from
lower surface 71 of lid 65 may be provided having an internal
diameter 74 slightly greater than the outer diameter of wells 64
thereby sealing wells 64 more effectively and reducing the
probability of cross-contamination of wells. It is to be
appreciated that in this configuration a resilient gasket is not
needed here for effective sealing. As an alternative to clips 72 as
shown in the figure, standard crocodile clips (not shown) could
also be used. It will be appreciated that the height, length and
width of the assembly 61 may be identical to the corresponding
dimensions of commercially available 96 well plates, so that
current instrumentation can still be used; also, it would still be
possible stack the assemblies on top of one another. Other
embodiments of the present invention can also be stacked. This
feature is also provided by the other techniques described herein
for clamping of lids on the sample containing plates/tube
arrays.
FIG. 4B shows a fragmentary perspective view of the clamping
mechanism described in detail with respect to FIG. 4. In FIG. 4B,
only projection 68 of plate 62 is shown; the rest of plate 62 is
not depicted. Each vertical male projection 68 preferably has
groove 76 which would engage clip 72 such that sufficient pressure
is applied to efficiently seal all the sample containment sites.
Alternative mechanisms of clips or clamping assemblies will be
apparent to those skilled in the art in light of the disclosure of
the present invention.
In another embodiment, and referring now to FIG. 5 of the drawings,
gasket 77 is shown as a discrete flat annular element inserted into
recess or annular bore or well 78 of plate 79. Plate 79 is shown as
a single well or tube unit broken out from the plate or tray.
Gasket 77 is disposed on shoulder 80 of well 78. Accordingly, lid
81 includes a projection 82 which fits into well 78 and mates with
gasket 77 on shoulder 80 when lid 81 is placed on plate 79 in the
proper orientation. When lid 81 is clamped in the proper
orientation on plate 79, gasket 77 is compressed between surface 83
of projection 82 and the shoulder 80 of well 78, thereby sealing
the well most preferably in a substantially hermetic manner. It is
to be understood, that other than those embodiments of the present
invention in which a gas permeable membrane is utilized, the seal
of the containment sites which is achieved will typically be an
hermetic seal. A similar arrangement is shown in FIG. 5A with two
modifications.
Referring now to FIG. 5A, the apparatus shown is an alternative
embodiment of that shown in FIG. 5. The resilient gasket comprises
an O-ring 84 which may rest on shoulder 85 or which may be disposed
in an annular channel 86 formed in shoulder 85, channel 86 being
shown in phantom. In this embodiment, lid 87 has a projection or
annular collar 88 with a central bore 89 such that only the surface
90 of collar 88 mates with O-ring 84 when lid 87 is placed on the
plate in the proper orientation. Lid 81 of FIG. 5 and lid 87 of
FIG. 5A are essentially interchangeable in FIG. 5 and FIG. 5A. Lid
87 may comprise a solid projection by simply filling in space 89
during the molding process. For the embodiments shown in FIG. 5 and
FIG. 5A, the assembly may be clamped in any suitable manner. FIG.
5B shows a plan view of an individual gasket or O-ring used in the
devices shown in FIG. 5 and FIG. 5A.
In still another embodiment, and referring now to FIG. 6 of the
drawings, assembly 91 is shown in fragmentary cross-sectional
elevational view, with one well of the plate broken away. It will
be appreciated that one of the important uses of this assembly will
be growth or processing of cell cultures, in which a high degree of
stringency is needed to prevent microbial, particulate or chemical
contamination while still allowing gas equilibration of the samples
with a controlled atmosphere, an example of which is 5 carbon
dioxide. Lid 92 is shown disposed on gas permeable membrane 93.
Membrane 93 is shown disposed on resilient gasket 94 having holes
95 in alignment with well openings 96 of wells 97 in plate 98. Lid
92 in this embodiment is not a single impermeable layer with a flat
principal surface an described above, but is formed such that it
has collars 99 projecting down from lid 92 with holes 204 in
collars 99. The lower surface 100 of collars 99 thus, press down on
membrane 93 when the apparatus is completely assembled. If so
desired, another similar resilient gasket (not shown) could be
placed between lid 92 and membrane 93. Also, membrane 93 could be
placed on a porous grid (not shown) or a thick filter paper sheet
to give mechanical support to membrane 93 with gasket 94 below the
mechanical support. Lid 92 would then be clamped by any of the
means previously described, lid 92 and plate 98 having been made
accordingly. By clamping the assembly in the correct format as
described, lower surfaces 100 of collars 99 would apply pressure to
the tops 101 of wells 97 via gasket 94, with the membrane 93
disposed between lid 92 and gasket 94, thereby sealing the wells
against particulate contamination and yet allowing gas
equilibration of samples in wells 97 through membrane 93 via holes
204. The membrane material is not critical, examples being
polycarbonate or polysulfone. It would be preferable to use a
hydrophobic (such as Teflon or PVDF) membrane, as this would serve
to prevent the flow-through of liquids through the membrane if the
plate were overturned; this would also be a distinct advantage when
hazardous materials are being processed. Thus, the preferred
membrane is a gas permeable/liquid impermeable membrane. The pore
size of the membrane is not critical; however, the pores should be
small enough to keep out micro-organisms, dust, etc. while allowing
free passage of gases. In a different embodiment, the membrane can
be an impermeable sheet (mylar) with holes in alignment with the
wells, with discs of gas-permeable membrane attached to the mylar
sheet such that the holes in the mylar sheet are covered. Thus, the
discs of gas-permeable membrane will be in alignment with the well
openings when the apparatus is completely assembled in the proper
manner. Variations of membrane material, pore size and type of
clipping mechanisms will be apparent to those skilled in the art
from the disclosure herein.
In another embodiment of the present invention, referring now to
FIG. 7 of the drawings, a single well broken away from the plate is
shown in fragmentary cross-sectional view. Lid 102 has annular
collar 103 protruding from it with membrane 104 fixed with
adhesive, solvent, heat or ultrasonic welding to the lower surface
105 of collar 103. Gasket 106 in this embodiment could be a flat
annular ring or an O-ring fixed to lower surface 105 of collar 103
with membrane disc 104 disposed in between. On clamping lid 102 in
place on plate 108, gasket or O-ring 106 would mate with shoulder
109 built in well 110 of plate 108. In an alternative embodiment,
gasket or O-ring 106 would be placed in an annular channel 111
built in shoulder 109 as shown in phantom, with annular collar 103
of lid 102 having only membrane 104 fixed to it. It is to be
appreciated that the lids used for the inventions described with
reference to FIGS. 6 and 7 would be very useful for tissue culture
of cells. Moreover, once the processing of the cells in a
controlled atmosphere is complete, the lids can be replaced with
the lids described earlier, i.e., where the sealing is achieved
with a gas impermeable seal; cells can thus be grown in the
apparatus shown in FIGS. 6 and 7, the lid replaced as described
above, and then frozen. Thus, the same plate can be used for tissue
culture as well as for subsequent freezing or hermetic sealing of
the cultures. Thus, this apparatus achieves a sort of hermetic
sealing of the samples by virtue of excluding all non-gaseous
matter from the wells while allowing gas exchange, and yet sealing
in all the non-gaseous sample in the well.
An alternative embodiment of the apparatuses shown in FIGS. 6 and 7
is shown in exploded perspective view in FIG. 7A. Plate 300 has a
gasket 301 which lies on the principal surface 302 of plate 300,
close to the edge of the plate. Membrane 303 lies on top of
principal surface 302 with gasket 301 disposed in between. Lid 304
in this embodiment is similar to lid 92 of FIG. 6, but also has a
wall 305 projecting down from the lower principal surface 306 along
the edge of the lid. Wall 305 would be placed such than when
assembled, the bottom of wall 306 would exert pressure on gasket
301 through membrane 303 thereby sealing the wells from external
contamination, while still allowing gas equilibration of the
samples in the wells. The membrane could also be fixed to the lower
principal surface 306 of lid 304 and covered with a porous grid for
mechanical support and for protecting the membrane from mechanical
damage.
Yet another embodiment of the present invention is shown in FIGS. 8
and 8A, where assembly 112 consists of tube tray 113 having
individual tubes 114. Tube tray 113 is held in place in tray holder
115 by means of male retaining arms 116 which mate with slots 117
in tray holder 115. When in place, the bottoms of tubes 114 will
project through holes 118 in tray holder 115. An enlarged,
fragmentary cross-sectional view of a single tube and well is given
in FIG. 8A. Cap cover assembly 122 is clamped down by lid 126 such
that caps 123 are in alignment with openings of tubes 114.
FIG. 8A shows an enlarged, cross-sectional fragmentary view of a
single tube 114 sealed by a cap 123 broken away from the tray and
cap assemblies. The top portions 119 of tubes 114 have flared necks
120 which rise significantly above the principal top surface 121 of
tube tray 113. The cap assembly 122 has individual caps 123 and
vertical walls 124 which project down vertically between caps 123
from the lower surface 125 of the cap assembly. Upon lid 126 being
clamped in place by any of the means previously described, the
walls 128 of caps 123 will mate with the flared necks 120 thereby
sealing tubes 114 with a friction fit between the two. Also, lower
surface 129 of vertical walls 124 will mate with the top surface
121 of tube tray 113. It will be obvious to those skilled in the
art, that as a result of the angle of projection of the cap walls
128, sealing is achieved with very little pressure. The method of
sealing here does not require tight fitting snap caps; thus, upon
removal of a clamp (not shown) lid 126 and cap assembly 122 will
rise a little bit because of the shape of walls 128 of caps 123,
thus providing some degree of rebound, without the use of a
rubber-like gasket material. However, even upon removal of the
clamp, walls 128 of individual caps 123 will still remain in place
mating with flared necks 120 of tubes 114, and the lower surface
129 of walls 124 will still extend significantly below the (top)
principal surface 130 of the tops 119 of tubes 114. Thus, the
chances of cross-contamination between individual tubes is
eliminated without the use of a resilient gasket as provided in
some of the previous embodiments. If so desired, the assembly 112
can be also designed such that when lid 126 is locked in place with
the clamp, the top surface 130 of tube neck 120 will also mate with
the (lower) principal surface 125 of lid 126, resulting in a third,
additional, sealing area and mechanism. Lid 126 in this embodiment
has holes 131 in the proper orientation such that when clamped in
place on tray carrier 115, the tops 132 of individual caps 123 will
project out above principal surface 133 of lid 126. This may be
advantageous in situations where uniform heating or cooling of
samples is required.
Finally, referring now to FIG. 9, another embodiment of the present
invention is shown in cross-sectional view. Slide 134 is shown
having a plurality of wells 135. Slide 134 also has shoulders 136
along the sides. Gasket 137 made of a resilient, rubber-like
material as previously described, in the form of a sheet, is shown
disposed around well 135 on slide 134. Cover 138 also has shoulders
139 corresponding to the positions of shoulders 136 of slide 134.
C-clamp 140 holds cover 138 in place on slide 134, pressure being
applied to gasket 137 by the lower surface 141 of cover 138,
thereby sealing wells 135. The gasket and cover may have a
non-symetrical geometry such that it may be placed on the slide
only in the correct orientation. Also, slide 134 need not have
shoulders 136; in this embodiment, they are provided such that a
majority of the lower principal surface of the slide is in contact
with the support on which it rests, ensuring uniform heating if
kept on a heating block. Shoulders 139 of cover 138 are provided
for the same purpose, so that the contents of the wells may be
uniformly heated from above. FIG. 9B9A shows a perspective view ofl
the apparatus described in detail with reference to FIG. 9. In FIG.
9A, gasket 137 and clamp 140 are not shown. An alternative
embodiment would provide gasket 143 in the form of an annular ring
or O-ring, similar to the gasket in FIG. 5B. Gasket 143 would rest
in an annular channel (not shown in FIG. 9 and FIG. 9A) formed
around each well. The internal diameter of the gasket would be
slightly smaller than the opening of the well, contributing to
sample confinement. As in earlier embodiments, a thermal
equilibrium membrane can also be used if desired. The gasket could
also be in the form of a substantially continuous sheet without
holes, as in some earlier embodiments. While the preferred
embodiment has been described in detail with respect to FIGS. 9 and
9A, another variation could be as follows. The slide assembly could
consist of a slide holder (comparable to the tray holder in FIG.
1), with the slide being in the form of a well-plate defining the
depressions or sample wells (similar to the tube tray in FIG. 1).
When the well-plate is in the proper orientation on top of the
slide holder, the wells of the well-plate would protrude from holes
in the slide holder (like the holes 17 in tray carrier 16 described
in FIG. 1). The lid could then be clamped to the slide holder with
the gasket in between the well-plate and the lid thereby sealing
the wells.
Thus, it is apparent that there has been provided in accordance
with the invention a method and apparatus that fully satisfies the
objects, aim and advantages set forth above. While the invention
has been described in connection with specific embodiments thereof,
it is evident that many alternatives, modifications, and variations
will be apparent to those skilled in the art in the light of the
foregoing description. Also, it is apparent that any of the
embodiments could be used with any other embodiment(s) depending on
the requirements. Accordingly, it is intended to embrace all such
alternatives, modifications and variations that fall within the
spirit and broad scope of the appended claims.
* * * * *